Glycerides are fatty acid esters of the triol glycerol. Specifically, triglycerides (or triacylglycerols), are fatty acid esters in which all three of the glycerol —OH groups have been esterified by fatty acids. Some of the many uses of triglycerides include utilization as a fat source in a variety of specialized nutritional products; carriers for flavors, vitamins, essential oils and colors; mineral oil alternatives; moisture barriers; clouding agents for beverages; primary and secondary emollients; lubricants; and solubilizing agents.
Particularly desirable to the nutrition and health-care fields are triglycerides of conjugated linoleic acid and conjugated linolenic acid (collectively known as “CLA”). CLAs have generated much interest in the academic and business communities because of their nutritional, therapeutic, and pharmacological properties. CLAs have become biologically and commercially important as they have been observed to inhibit mutagenesis and to provide unique nutritional value. Additionally, CLAs promote body fat reduction, body weight reduction, increased muscle mass, increased feed efficiency, prevention of weight loss due to immune stimulation, elevated CD-4 and/or CD-8 cell counts in animals, increased bone mineral content, prevention of skeletal abnormalities in animals, and/or decreased blood cholesterol levels. Due to the natural esterases found in mammals, the CLA-ester may be readily cleaved to release the desirable free fatty acid. Therefore, CLA triglycerides are desirable since they are much more stable to oxidation than free fatty acids, thus lending to a longer product shelf-life. Furthermore, CLA glycerides are increasingly fat miscible with increasing CLA acylation. See, e.g., U.S. Pat. No. 6,136,985 (Millis) and WO 00/18944 (Conlinco, Inc.) for discussion of CLA esters and uses thereof. Besides CLAs, however, desirable acyl group donors for production of triglycerides may include other free fatty acids and fatty acid derivatives containing from about 2 to 24 carbon atoms.
Glyceride esters of fatty acids are generally prepared by an esterification reaction of glycerol with a corresponding fatty acid, an alcohol interchange reaction of glycerol with oil or fat, or other similar reactions. The reaction processes can be divided into two groups: chemical reactions, which utilize an acidic or alkali catalyst or the like, and biochemical processes, which utilize fat-hydrolyzing enzymes.
In chemical processes, generally, the first and third hydroxyl positions of the glycerol molecule are acylated first, while the second position is later acylated. This type of reaction to completion is difficult and time-consuming, however. A variety of chemical processes used to prepare esters are generally known to those skilled in the art. These methods include acid-catalyzed reaction of acids and alcohols, alkali-catalyzed transesterification of acyl esters with alcohols, and the like.
An alternative to chemical methodology is the utilization of fat-hydrolyzing enzymes such as various lipases. Fatty acids and/or fatty acid derivatives can be reacted in the presence of solid phase bound lipases. WO 91/16443 (NovoNordisk AS), for example, discloses a method utilizing Candida antarctica lipase, Candida fugosa lipase, and other enzymes to catalyze formation of triglycerides from fatty acids or their derivatives in combination with glycerol. U.S. Pat. No. 6,361,980 (Sugiura) describes the enzymatic production of 1,3 diglycerides. WO 0018944 (Conlinco, Inc.), WO 9932105 (DCV, Inc.), and U.S. Pat. No. 6,136,985 (Millis) describe the use of enzymes to esterify CLA while GB 1,577,933 describes the use of enzymes to interesterify and incorporate fatty acids into pre-existing triglycerides.
However, the previous processes described above utilize a reaction chamber maintained at a temperature conducive to enzymatic catalyzation. These processes provide no mechanism for isomerization of the 1,3-diglycerides to 1,2-diglycerides. Specifically, no separate higher temperature thermal rearrangement zone is provided.
At temperatures conducive to enzymatic catalysis, meanwhile, the 1,3 selective lipase generally used will esterify the first and third hydroxyl positions first. Further reaction from 1,3 diglycerides to triglycerides will only proceed at a very slow rate, ill-suited to industrial application. Those skilled in the art will recognize that a 1,3 specific lipase has generally not been suitable for catalyzing triglyceride synthesis. See, e.g., F. Ergan et al., “Effect of Lipase Specificity on Trigylceride Synthesis,” 13 Biotech. Letters, No. 1, pp. 19-24 (1991) (“Ergan et al.”). Ergan et al. states that conventional methods for production of triglycerides using enzymatic catalyzation either produce low triglyceride yield or require very long reaction time, i.e. 6 weeks in some instances. Specifically, Ergan et al. recognize that a means of catalyzing 1,3 diglyceride to 1,2 diglyceride isomerization has been long-felt and required to effectively and efficiently produce triglycerides, but offers no resolution for such a need.
Thus, there is presently a need for a more efficient and industrially practical process for the production of triglycerides, including conjugated linoleic and linolenic acid triglycerides, utilizing at least one enzymatic catalysis zone containing 1,3 specific lipase and at least one thermal rearrangement zone to isomerize 1,3 diglycerides to 1,2 diglycerides to efficiently produce triglycerides, including conjugated linoleic acid triglycerides.